Project Summary: The objective of this research is to develop a new generation of fusion proteins that can be used for improved imaging and therapy of cancer and other diseases by the method of pretargeting, which separates antibody localization from the delivery of diagnostic or therapeutic radionuclides. This application describes a novel technology for the facile and quantitative assembly of trivalent, bispecific, binding structures from 2 distinct fusion proteins, and proposes to advance prototype molecules composed of three Fab fragments that are stably linked and retain the binding affinity of the parental antibodies. These novel constructs were purified to homogeneity and shown to be useful for pretargeted delivery of radio-labeled tracers to human colonic tumor xenografts in nude mice, achieving exceptional tumor-to-nontumor ratios. For example, at 1h post-injection of a 99mTc-labeled tracer, the tumor to blood ratio was as high as 66+5, with tumor uptake exceeding 30% injected dose per gram. The primary aim of this application is to further characterize 1 such construct both in vitro and in vivo, so that the feasibility of its future clinical development for pretargeted therapy can be defined. Another key aim is to show that this new construct can be prepared more efficiently and with higher productivity than previously-generated recombinant bispecific antibodies from this group. Upon meeting these milestones, we should be in a position to submit a Phase II application directed toward additional development of this agent for initial clinical testing as pretargeted radioimmunotherapy of CEA-positive cancers, such as colonic cancer. Project Narrative: This innovative research will produce multivalent, multifunctional, structures in general, and trivalent bispecific Fab-based binding proteins in particular, that can be useful for the detection, imaging, and therapy of various human diseases which have antigen targets, including cancers, cardiovascular diseases, autoimmune diseases, and infectious diseases. [unreadable] [unreadable] [unreadable]